Smad6 and uses thereof

ABSTRACT

The invention includes nucleic acids encoding the Smad6 protein, including fragments and biologically functional variants thereof. Also included are polypeptides and fragments thereof encoded by such nucleic acids, and antibodies relating thereto. Methods and products for using such nucleic acids and polypeptides also are provided.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/923,922,filed Aug. 7, 2001, now U.S. Pat. No. 6,534,476, issued Mar. 18, 2003,which is a divisional of application Ser. No. 09/096,776, filed Jun. 12,1998, now U.S. Pat. No. 6,270,994, issued Aug. 7, 2001, which claimspriority under 35 U.S.C. §119(e) from U.S. provisional application Ser.No. 60/049,990, filed Jun. 13, 1997, from U.S. provisional applicationSer No. 60/053,040, filed Jul. 18, 1997, and from U.S. provisionalapplication Ser. No. 60/066,173, filed Nov. 18, 1997.

FIELD OF THE INVENTION

This invention relates to nucleic acids and encoded polypeptides whichinteract with the TGF-β superfamily receptor complexes and which are anegative regulators of TGF-β superfamily signalling. The invention alsorelates to agents which bind the nucleic acids or polypeptides. Theinvention further relates to methods of using such nucleic acids andpolypeptides in the treatment and/or diagnosis of disease.

BACKGROUND OF THE INVENTION

During mammalian embryogenesis and adult tissue homeostasis transforminggrowth factor β (TGF-β) performs pivotal tasks in intercellularcommunication (Roberts et al., Growth Factors 8:1-9, 1993). The cellulareffects of this pleiotropic factor are exerted by ligand-inducedhetero-oligomerization of two distantly related type I and type IIserine/threonine kinase receptors, TβR-I and TβR-II, respectively (Linand Lodish, Trends Cell Biol. 11:972-978, 1993; Derynck, Trends Biochem.Sci. 19-:548-553, 1994; Massague and Weis-Garcia, Cancer Surv. 27:41-64,1996; ten Dijke et al., Curr. Opin. Cell. Bio. 8:139-145, 1996). The tworeceptors, which both are required for signalling, act in sequence;TβR-I is a substrate for the constitutively active TβR-II kinase (Wranaet al., Nature 370:341-347, 1994; Weiser et al., EMBO J. 14:2199-2208,1995).

TGF-β is the prototype of a large family of structurally relatedproteins that are involved in various biological activities (Massagué,et al., Trends Cell Biol. 7:187-192, 1997; Roberts & Sporn, in: Peptidegrowth factors and their receptors, Part I (Sporn, M. B. and Roberts, A.B., eds) pp. 319-472, Springer-Verlag, Heidelberg (1990); Yingling etal., Biochim. Biophys. Acta 1242:115-136, 1995). The TGF-β “superfamily”includes activins and bone morphogenetic proteins (BMPs) that signal ina similar fashion, each employing distinct complexes of type I and typeII serine/threonine kinase receptors (Lin and Lodish, 1993; Derynck,1994; Massague and Weis-Garcia, 1996; ten Dijke et al., 1996). TGF-βrelated molecules act in environments where multiple signals interactand are likely to be under tight spatial and chronological regulation.For example, activin and BMP exert antagonistic effects in thedevelopment of Xenopus embryos (Graff et al., Cell 85:479-487, 1996).Chordin (Piccolo et al., Cell 86:589-598, 1996) and noggin (Zimmerman etal., Cell 86:599-606, 1996), for example, inhibit the ventralizingeffect of BMP4 by binding specifically to the ligand. Likewise,follistatin neutralizes the activity of activin (Hemmati-Brivalou etal., Cell 77:283-295, 1994).

Genetic studies of TGF-β-like signalling pathways in Drosophila andCaenorhabditis elegans have led to the identification of mothers againstdpp (Mad) (Sekelsky et al., Genetics 139:1347-1358, 1995) and sma(Savage et al., Proc. Natl. Acad. Sci. USA 93:790-794, 1996) genes,respectively. The products of these related genes perform essentialfunctions downstream of TGF-β-like ligands acting via serine/threoninekinase receptors in these organisms (Wiersdorff et al., Development122:2153:2163, 1996; Newfeld et al., Development 122:2099-2108, 1996;Hoodless et al., Cell 85:489-500, 1996). Vertebrate homologs of Mad andsma have been termed Smads (Derynck et al., Cell 87:173, 1996) or MADRgenes (Wrana and Attisano, Trends Genet. 12:493-496, 1996). Geneticalterations in Smad2 and Smad4/DPC4 have been found in specific tumorsubsets, and thus Smads may function as tumor suppressor genes (Hahn etal., Science 271:350-353, 1996; Riggins et al., Nature Genet.13:347-349,1996; Eppert et al., Cell 86:543-552, 1996). Smad proteinsshare two regions of high similarity, termed MH1 and MH2 domains,connected with a variable proline-rich sequence (Massague, Cell85:947-950, 1996; Derynck and Zhang, Curr. Biol. 6:1226-1229, 1996). TheC-terminal part of Smad2, when fused to a heterologous DNA-bindingdomain, was found to have transcriptional activity (Liu et al., Nature381:620-623, 1996; Meersseman et al., Mech. Dev. 61:127-1400, 1997). Theintact Smad2 protein when fused to a DNA-binding domain,-was latent, buttranscriptional activity was unmasked after stimulation with ligand (Liuet al., 1996).

Different Smads specify different responses using functional assays inXenopus. Whereas Smad1 induces ventral mesoderm, a BMP-like response,Smad2 induces dorsal mesoderm, an activin/TGF-β-like response (Graff etal., Cell 85:479-487, 1996; Baker and Harland, Genes & Dev.10:1880-1889, 1996; Thomsen, Development 122:2359-2366, 1996). Uponligand stimulation Smads become phosphorylated on serine and threonineresidues; BMP stimulates Smad1 phosphorylation, whereas TGF-β inducesSmad2 and Smad3 phosphorylation (Hoodless et al., Cell 85:489-500, 1996;Liu et al., 1996; Eppert et al., 1996; Lechleider et al., J. Biol. Chem.271:17617-17620, 1996; Yingling et al., Proc. Nat'l Aced. Sci.USA93:8940-8944, 1996; Zhang et al., Nature 383:168-172, 1996;Macías-Silva et al., Cell 87:1215-1224, 1996; Nakao et al., J. Biol.Chem. 272:2896-2900, 1996).

Smad4 is a common component of TGF-β, activin and BMP signalling (Lagnaet al., Nature 383:832-836, 1996; Zhang et al., Curr. Biol. 7:270-276,1997; de Winter et al., Oncogene 14:1891-1900, 1997). Smad4phosphorylation has thus far been reported only after activinstimulation of transfected cells (Lagna et al., 1996). After stimulationwith TGF-β or activin Smad4 interacts with Smad2 or Smad3, and upon BMPchallenge a heteromeric complex of Smad4 and Smad1 has been observed(Lagna et al., 1996). Upon ligand stimulation, Smad complexestranslocate from the cytoplasm to the nucleus (Hoodless et al., 1996;Liu et al., 1996; Baker and Harland, 1996; Macías-Silva et al., 1996),where they, in combination with DNA-binding proteins, may regulate genetranscription (Chen et al., Nature 383:691-696, 1996).

SUMMARY OF THE INVENTION

The invention provides isolated nucleic acid molecules, unique fragmentsof those molecules, expression vectors containing the foregoing, andhost cells transfected with those molecules. The invention also providesisolated polypeptides and agents which bind such polypeptides, includingantibodies. The foregoing can be used in the diagnosis or treatment ofconditions characterized by the expression of a Smad6 nucleic acid orpolypeptide, or lack thereof. The invention also provides methods foridentifying pharmacological agents useful in the diagnosis or treatmentof such conditions. Here, we present the identification of Smad6, whichinhibits phosphorylation of pathway specific Smad polypeptides includingSmad2 and Smad1 and inhibits the TGF-β superfamily signalling pathwaysuch as the TGF-β and BMP signalling pathways.

According to one aspect of the invention, an isolated nucleic acidmolecule is provided. The molecule hybridizes under stringent conditionsto a molecule consisting of the nucleic acid sequence of SEQ ID NO: 1.The isolated nucleic acid molecule codes for a polypeptide whichinhibits TGF-β, activin, or BMP signalling. The invention furtherembraces nucleic acid molecules that differ from the foregoing isolatednucleic acid molecules in codon sequence due to the degeneracy of thegenetic code. The invention also embraces complements of the foregoingnucleic acids.

In certain embodiments, the isolated nucleic acid molecule comprises amolecule consisting of the nucleic acid sequence of SEQ ID NO: 3 orconsists essentially of the nucleic acid sequence of SEQ ID NO: 1.Preferably, the isolated nucleic acid molecule consists of the nucleicacid sequence of SEQ ID NO: 3,

According to another aspect of the invention, an isolated nucleic acidmolecule is provided. The isolated nucleic acid molecule comprises amolecule consisting of a unique fragment of SEQ ID NO: 3 between 12 and1487 nucleotides in length and complements thereof, provided that theisolated nucleic acid molecule excludes sequences consisting only of SEQID NO: 4. In one embodiment, the isolated nucleic acid molecule consistsof between 12 and 32 contiguous nucleotides of SEQ ID NO: 1, orcomplements of such nucleic acid molecules. In preferred embodiments,the unique fragment is at least 14, 15, 16, 17, 18, 20 or 22 contiguousnucleotides of the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3,or complements thereof.

According to another aspect of the invention, the invention involvesexpression vectors, and host cells transformed or transfected with suchexpression vectors, comprising the nucleic acid molecules describedabove.

According to still other aspects of the invention, transgenic non-humananimals are provided. The animals include in certain embodiments theforegoing expression vectors. In certain preferred embodiments, thetransgenic non-human animal includes a conditional Smad6 expressionvector, such as an expression vector that increases expression of Smad6in a tissue specific, development stage specific, or inducible manner.In other embodiments, the transgenic non-human animal has reducedexpression of Smad6 nucleic acid molecules. In some embodiments, thetransgenic non-human animal includes a Smad6 gene disrupted byhomologous recombination. The disruption can be homozygous orheterozygous. In other embodiments, the transgenic non-human animalincludes a conditional Smad6 gene disruption, such as one mediated bye.g. tissue specific, development stage specific, or inducible,expression of a recombinase. In yet other embodiments, the transgenicnon-human animal includes a trans-acting negative regulator of Smad6expression, such as antisense Smad6 nucleic acid molecules, nucleic acidmolecules which encode dominant negative Smad6 proteins, Smad6 directedribozymes, etc.

According to another aspect of the invention, an isolated polypeptide isprovided. The isolated polypeptide is encoded by the isolated nucleicacid molecule of any of claims 1, 2, 3, or 4, and the polypeptide hasTGF-β, activin, or BMP signalling inhibitory activity. Preferably, theisolated polypeptide consists-of the amino acid sequence of SEQ ID NO:2.

In other embodiments, the isolated polypeptide consists of a fragment orvariant of the foregoing which retains the activity of the foregoing.

According to another aspect of the invention, there are providedisolated polypeptides which selectively bind a Smad6 protein or fragmentthereof, provided that the isolated polypeptide is not a TGF-βsuperfamily receptor, such as a TGF-β, activin or BMP type I receptor.The isolated polypeptide in certain embodiments binds to a polypeptideencoded by the isolated nucleic acid molecule of any of claims 1, 2, 3,or 4. Preferred isolated polypeptides bind to an epitope defined by apolypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Inother preferred embodiments, isolated binding polypeptides includeantibodies and fragments of antibodies (e.g., Fab, F(ab)₂, Fd andantibody fragments which include a CDR3 region which binds selectivelyto the Smad6 polypeptides of the invention). In still other preferredembodiments, the isolated polypeptide is a monoclonal antibody, ahumanized antibody or a chimeric antibody.

The invention provides in another aspect an isolated complex ofpolypeptides. The isolated complex includes a TGF-β superfamily receptoror receptor complex bound to a polypeptide as claimed in claim 16.Preferably the isolated complex comprises a polypeptide having the aminoacid sequence of SEQ ID NO: 2. In other preferred embodiments, thereceptor or receptor complex is selected from the group consisting ofTβRI, BMPR-IA, BMPR-IB, ActR-IA, a complex of TβRI and TβRII, a complexof BMPR-IA and BMPR-II, a complex of BMPR-IB and BMPR-II, a complex ofActR-IA and BMPR-II and a complex of ActR-IA and ActR-II.

According to still another aspect of the invention, methods for reducingTGF-β superfamily signal transduction in a mammalian cell are provided.The methods involve contacting a mammalian cell with an amount of aninhibitor of TGF-β superfamily signal transduction effective to reducesuch signal transduction in the mammalian cell. Preferably the TGF-βsuperfamily signal transduction is mediated by a TGFβ superfamilyligand, particularly TGF-β1, activin, Vg1, BMP-4 and/or BMP-7. Othermethods are provided for modulating phosphorylation of pathway specificSmads (e.g. Smad1, Smad2, Smad3 and/or Smad5). Certain methods areprovided for reducing phosphorylation of Smad1 or Smad2 in a mammaliancell by contacting the mammalian cell with an agent which reduces Smad1or Smad2 phosphorylation, respectively. Still other methods are providedfor increasing phosphorylation of Smad3 in a mammalian cell bycontacting the mammalian cell with an agent which increases Smad3phosphorylation. In certain embodiments of the foregoing methods, theagent is an isolated Smad6 polypeptide, such as a polypeptide encoded bya nucleic acid which hybridizes under stringent conditions or thenucleic acid of SEQ ID NO: 1, or degenerates or complements thereof.

According to still another aspect of the invention, methods formodulating proliferation and/or differentiation of a cancer cell areprovided. The methods involve contacting a cancer cell with an amount ofan isolated Smad6 polypeptide as described above, effective to reduceproliferation and/or differentiation of the cancer cell.

The invention in a further aspect provides methods for increasing TGF-βsuperfamily signal transduction in a mammalian cell. The mammalian cellis contacted with an agent that selectively binds to an isolated nucleicacid molecule of the invention or an expression product thereof in anamount effective to increase TGF-β superfamily signal transduction.Preferably the TGF-β superfamily signal transduction is mediated by aTGFβ superfamily ligand selected from the group consisting of TGF-β1,activin, Vg1, BMP-4 and BMP-7. Preferred agents are antisense Smad6nucleic acids, including modified nucleic acids, and polypeptidesincluding antibodies which bind to a Smad6 polypeptide including theamino acids of SEQ ID NO: 2, and a dominant negative variant of thepolypeptide of SEQ ID NO: 2.

The invention in still another aspect provides compositions comprising aSmad6 polypeptide and a pharmaceutically acceptable carrier.

The invention in a further aspect involves a method for decreasing Smad6TGF-β superfamily inhibitory activity in a subject. An agent thatselectively binds to an isolated nucleic acid molecule of the inventionor an expression product thereof is administered to a subject in need ofsuch treatment, in an amount effective to decrease TGFβ superfamilysignal transduction inhibitory activity of Smad7 in the subject.Preferably the TGFβ superfamily signal transduction is mediated by aTGFβ superfamily ligand selected from the group consisting of TGF-β1,activin, Vg1, BMP-4 and BMP-7. Preferred agents are antisense nucleicacids, including modified nucleic acids, and polypeptides includingantibodies which bind to the polypeptide including the amino acids ofSEQ ID NO: 2, and dominant negative variants of the polypeptide of SEQID NO: 2.

According to yet another aspect of the invention, methods for treating acondition characterized by abnormal BMP activity are provided. Themethods include administering to a subject in need of such treatment aneffective amount of Smad6 or a Smad6 agonist or antagonist sufficient torestore the BMP activity to normal. In some embodiments, the conditionis selected from the group consisting of ossification of the posteriorlongitudinal ligament and ossification of the ligament flavum.

According to another aspect of the invention, methods for treating acondition characterized by abnormal TGF-β activity are provided. Themethods include administering to a subject in need of such treatment aneffective amount of Smad6 or a Smad6 agonist or antagonist sufficient torestore the TGF-β activity to normal. In certain embodiments, thecondition is selected from the group consisting of liver fibrosisincluding cirrhosis and veno-occlusive disease; kidney fibrosisincluding glomerulonephritis, diabetic nephropathy, allograft rejectionand HIV nephropathy; lung fibrosis including idiopathic fibrosis andautoimmune fibrosis; skin fibrosis including systemic sclerosis,keloids, hypertrophic burn scars and eosinophilia-myalgia syndrome;arterial fibrosis including vascular restenosis and atherosclerosis;central nervous system fibrosis including intraocular fibrosis; andother fibrotic diseases including rheumatoid arthritis and nasalpolyposis.

In another aspect of the invention, methods for modulating theexpression of cyclin A are provided. The methods include contacting acell with Smad6 or an agonist or antagonist thereof, in an amounteffective to modulate the expression of cyclin A. In some embodimentsthe cell is contacted with Smad6, and the expression of cyclin A isincreased. In other embodiments, the cell is contacted with anantagonist of Smad6, and the expression of cyclin A is decreased.Preferably the antagonist of Smad6 is selected from the group consistingof antibodies to Smad6, dominant negative variants of Smad6 and Smad6antisense nucleic acids.

According to another aspect of the invention, methods are provided foridentifying lead compounds for a pharmacological agent useful in thediagnosis or treatment of disease associated with Smad6 TGF-βsuperfamily signal transduction inhibitory activity. One set of methodsinvolves forming a mixture of a Smad6 polypeptide, a TGF-β superfamilyreceptor or receptor complex, and a candidate pharmacological agent. Themixture is incubated under conditions which, in the absence of thecandidate pharmacological agent, permit a first amount of specificbinding of the TGF-β superfamily receptor or receptor complex by theSmad6 polypeptide. A test amount of the specific binding of the TGF-βsuperfamily receptor or receptor complex by the Smad6 polypeptide thenis detected. Detection of an increase in the foregoing activity in thepresence of the candidate pharmacological agent indicates that thecandidate pharmacological agent is a lead compound for a pharmacologicalagent which increases the Smad6 TGF-β superfamily signal transductioninhibitory activity. Detection of a decrease in the foregoing activitiesin the presence of the candidate pharmacological agent indicates thatthe candidate pharmacological agent is a lead compound for apharmacological agent which decreases Smad6 TGF-β superfamily signaltransduction inhibitory activity. Another set of methods involvesforming a mixture as above, adding further a pathway specific Smadpolypeptide, and detecting first and test amounts of TGF-β superfamilyinduced phosphorylation of the pathway specific Smad polypeptide.Detection of an increase in the phosphorylation in the presence of thecandidate pharmacological agent indicates that the candidatepharmacological agent is a lead compound for a pharmacological agentwhich decreases the Smad6 TGF-β superfamily signal transductioninhibitory activity. Detection of a decrease in the foregoing activitiesin the presence of the candidate pharmacological agent indicates thatthe candidate pharmacological agent is a lead compound for apharmacological agent which increases Smad6 TGF-β superfamily signaltransduction inhibitory activity. Preferred Smad6 polypeptides includethe polypeptides of claim 16. Preferably the TGFβ superfamily receptoris selected from the group consisting of TGFβ superfamily type Ireceptors, TGFβ superfamily type II receptors, and complexes of TGFβsuperfamily type I receptors and TGFβ superfamily type II receptors.Preferred pathway specific Smad polypeptides include Smad1 and Smad2.

According to still another aspect of the invention, methods forincreasing phosphorylation of Smad3 in a mammalian cell are provided.The methods include contacting the mammalian cell with an amount of anisolated Smad6 polypeptide effective to increase phosphorylation ofSmad3 in the mammalian cell.

According to another aspect of the invention, methods for reducingheteromerization of Smad2 with Smad3 or Smad4 in a mammalian cell areprovided. The methods include contacting the mammalian cell with anamount of an isolated Smad6 nucleic acid or polypeptide, or an agonistthereof, effective to reduce heteromerization of Smad2 with Smad3 orSmad4 in the mammalian cell.

The use of the foregoing compositions, nucleic acids and polypeptides inthe preparation of medicaments also is provided.

In the foregoing compositions and methods, preferred members of theTGF-β superfamily are TGF-β1, activin, Vg1, BMP-4 and BMP-7, and thepreferred pathway specific Smad polypeptides are Smad1, Smad2, Smad3 andSmad5.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1 to 1A-3 and 1B are a representation of a photograph whichdepicts (A) the protein sequence alignments of Smad6 (SEQ ID NO: 2) withSmads 1-5 (SEQ ID NOs: 6-10) and (B) the tissue distribution of mouseand human Smad6 in human (left) and a mouse (right) tissue blots.

FIGS. 2 a to 2 c is a representation of a photograph which shows thebinding of Smad6 to type I receptors; (A) TβR-I, (B) ActR-IB, (C)BMPR-IB.

FIG. 3 (panels A-D) is a representation of a photograph whichdemonstrates the effect of Smad6 on the phosphorylation of Smad1, Smad 2and Smad3.

FIG. 4 (panels A-C) is a representation of a photograph whichdemonstrates the effect of Smad6 on the heteromerization of Smad2, Smad3and Smad4.

FIG. 5 (panels A-C) is a representation of a photograph which shows theeffect of Smad6 on transcriptional responses of TGF-β.

FIG. 6 (panels A-C) is a representation of a photograph which shows theexpression of Smad6 following stimulation of cells with BMP-2 (A),BMP-7/OP-1 (B) or TGF-β1 (C).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the nucleotide sequence of the mouse Smad6 cDNA.

SEQ ID NO: 2 is the amino acid sequence of the mouse Smad6 protein.

SEQ ID NO: 3 is the nucleotide sequence of the coding region of themouse Smad6 cDNA.

SEQ ID NO: 4 is the nucleotide sequence of the Smad6-related cDNA havingGenBank accession number U59914.

SEQ ID NO: 5 is the amino acid sequence of the Smad6-related proteinhaving GenBank accession number U59914.

SEQ ID NO: 6 is the amino acid sequence of the Smad1 protein, shown inFIG. 1.

SEQ ID NO: 7 is the amino acid sequence of the Smad2 protein, shown inFIG. 1.

SEQ ID NO: 8 is the amino acid sequence of the Smad3 protein, shown inFIG. 1.

SEQ ID NO: 9 is the amino acid sequence of the Smad4 protein, shown inFIG. 1.

SEQ ID NO: 10 is the amino acid sequence of the Smad5 protein, shown inFIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

It has previously been shown that Smad1, Smad2, Smad3, and Smad5transduce ligand-specific signals. In addition, Smad4 acts as anessential common partner of these ligand-specific Smads. In the presentwork, Smad6 is reported as a member belonging to a third class of theSmad family. Smad6 has a quite distinct structure from the Smad1, Smad2,Smad3, Smad4 and Smad5, and is believed to be a negative regulator insignaling of the TGF-β superfamily. Although not wishing to be bound bya precise mechanism, it is believed that the regulatory step by Smad6 isat the receptor level since Smad6 stably associates with the type Ireceptors.

The present invention in one aspect involves the cloning of a cDNAencoding a Smad6 protein which interacts with TGF-β superfamilyreceptors. The TGF-β superfamily members are well known to those ofordinary skill in the art and include TGF-βs, activins, bonemorphogenetic proteins (BMPs), Vg1, Mullerian inhibitory substance (MIS)and growth/differentiation factors (GDFs). The sequence of the mouseSmad6 gene is presented as SEQ ID NO: 1, and the predicted amino acidsequence of this gene's protein product is presented as SEQ ID NO: 2.Analysis of the sequence by comparison to nucleic acid and proteindatabases determined that Smad6 has a C-terminal domain (the MH2 domain)which is related to other Smad proteins (FIG. 1).

The invention thus involves in one aspect Smad6 polypeptides, genesencoding those polypeptides, functional modifications and variants ofthe foregoing, useful fragments of the foregoing, as well astherapeutics relating thereto.

Homologs and alleles of the Smad6 nucleic acids of the invention can beidentified by conventional techniques. For example, the human homolog ofSmad6 can be isolated by hybridizing a probe derived from SEQ ID NO: 1under stringent conditions a human cDNA library and selecting positiveclones. The existence, size, and tissue distribution of a human homologis demonstrated in the examples by Northern blot. Thus, an aspect of theinvention is those nucleic acid sequences which code for Smad6polypeptides and which hybridize to a nucleic acid molecule consistingof SEQ ID NO: 1, under stringent conditions. The term “stringentconditions” as used herein refers to parameters with which the art isfamiliar. Nucleic acid hybridization parameters may be found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. More specifically, stringentconditions, as used herein, refers, for example, to hybridization at 65°C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS,2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH7; SDSis sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed at 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS attemperatures up to 65° C.

There are other conditions, reagents, and so forth which can used, whichresult in a similar degree of stringency. The skilled artisan will befamiliar with such conditions, and thus they are not given here. It willbe understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof homologs and alleles of Smad6 nucleic acids of the invention. Theskilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

In general, homologs and alleles typically will share at least 40%nucleotide identity and/or at least 50% amino acid identity to SEQ IDNO: 1 and SEQ ID NO: 2, respectively, in some instances will share atleast 50% nucleotide identity and/or at least 65% amino acid identityand in still other instances will share at least 60% nucleotide identityand/or at least 75% amino acid identity. Watson-Crick complements of theforegoing nucleic acids also are embraced by the invention.

In screening for Smad6 proteins, a Southern blot may be performed usingthe foregoing conditions, together with a radioactive probe. Afterwashing the membrane to which the DNA is finally transferred, themembrane can be placed against X-ray film to detect the radioactivesignal.

The invention also includes degenerate nucleic acids which includealternative codons to those present in the native materials. Forexample, serine residues are encoded by the codons TCA, AGT, TCC, TCG,TCT and AGC. Each of the six codons is equivalent for the purposes ofencoding a serine residue. Thus, it will be apparent to one of ordinaryskill in the art that any of the serine-encoding nucleotide triplets maybe employed to direct the protein synthesis apparatus, in vitro or invivo, to incorporate a serine residue into an elongating Smad6polypeptide. Similarly, nucleotide sequence triplets which encode otheramino acid residues include, but are not limited to: CCA, CCC, CCG andCCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons);ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparaginecodons); and ATA, ATC and ATT (isoleucine codons). Other amino acidresidues may be encoded similarly by multiple nucleotide sequences.Thus, the invention embraces degenerate nucleic acids that differ fromthe biologically isolated nucleic acids in codon sequence due to thedegeneracy of the genetic code.

The invention also provides isolated unique fragments of SEQ ID NO: 1 orcomplements of SEQ ID NO: 1. A unique fragment is one that is a‘signature’ for the larger nucleic acid. It, for example, is long enoughto assure that its precise sequence is not found in molecules outside ofthe Smad6 nucleic acids defined above. Unique fragments can be used asprobes in Southern blot assays to identify such nucleic acids, or can beused in amplification assays such as those employing PCR. As known tothose skilled in the art, large probes such as 200 nucleotides or moreare preferred for certain uses such as Southern blots, while smallerfragments will be preferred for uses such as PCR. Unique fragments alsocan be used to produce fusion proteins for generating antibodies ordetermining binding of the polypeptide fragments, as demonstrated in theExamples, or for generating immunoassay components. Likewise, uniquefragments can be employed to produce nonfused fragments of the Smad6polypeptides, useful, for example, in the preparation of antibodies, inimmunoassays, and as a competitive binding partner of the TGF-βreceptor, activin receptor or BMP receptor and/or other polypeptideswhich bind to the Smad6 polypeptides, for example, in therapeuticapplications. Unique fragments further can be used as antisensemolecules to inhibit the expression of Smad6 nucleic acids andpolypeptides, particularly for therapeutic purposes as described ingreater detail below.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NO: 1 and its complement will requirelonger segments to be unique while others will require only shortsegments, typically between 12 and 32 nucleotides (e.g. 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32bases long). Excluding nucleic acid molecules consisting completely ofthe nucleotide sequence of SEQ ID NO: 4 or fragments thereof (GenBankaccession number U59914) which overlap SEQ ID NO: 1, virtually anysegment of SEQ ID NO: 1, or complements thereof, that is 18 or morenucleotides in length will be unique. A fragment which is completelycomposed of the sequence of SEQ ID NO: 4 or fragments thereof is onewhich does not include any of the nucleotides unique to Smad6. Preferredlonger unique fragments include those which are at least 35, 40, 45, 50,55, 60, 65, 70, 75, 100, 150, 200, 250, 300, or 500 nucleotide inlength. Particularly preferred are those unique fragments drawncompletely from the portion of SEQ ID NO: 3 which is not overlapped bySEQ ID NO: 4.

The unique fragments of the invention exclude sequences identical withcertain particular prior art nucleic acids or that are identical to onlyfragments thereof It is intended that the claims not embrace suchmolecules which are in the prior art. For example, portions of prior artESTs having GenBank accession numbers AA451501, AA046702, W72479,AA131352, AA131266, W41111, N48277, and the like, which are identical tothe Smad6 sequence of the invention are not unique fragments of Smad6.Thus, the nucleic acids which consist only of these sequences, or whichconsist only of fragments of these sequences, are considered to bewithin the prior art. Nucleic acids, however, which include any portionof the novel sequence of the invention are embraced by the invention,including sequences comprising contiguous portions of the novelsequences and the prior art sequences. Such sequences have unexpectedproperties, as described herein. In one embodiment the unique fragmentdoes not include any portion of the excluded prior art sequences. Thoseskilled in the art are well versed in methods for selecting suchsequences, typically on the basis of the ability of the unique fragmentto selectively distinguish the sequence of interest from non-Smad6nucleic acids. A comparison of the sequence of the fragment to those onknown data bases typically is all that is necessary, although in vitroconfirmatory hybridization and sequencing analysis may be performed.

A unique fragment can be a functional fragment. A functional fragment ofa nucleic acid molecule of the invention is a fragment which retainssome functional property of the larger nucleic acid molecule, such ascoding for a functional polypeptide, binding to proteins, regulatingtranscription of operably linked nucleic acids, and the like. One ofordinary skill in the art can readily determine using the assaysdescribed herein and those well known in the art to determine whether afragment is a functional fragment of a nucleic acid molecule using nomore than routine experimentation.

As mentioned above, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a Smad6polypeptide, to increase TGF-β superfamily signalling by reducing theamount of Smad6. This is desirable in virtually any medical conditionwherein a reduction of Smad6 is desirable, e.g., to increase TGF-βsignalling.

As used herein, the term “antisense oligonucleotide” or “antisense”describes an oligonucleotide that is an oligoribonucleotide,oligodeoxyribonucleotide, modified oligoribonucleotide, or modifiedoligodeoxyribonucleotide which hybridizes under physiological conditionsto DNA comprising a particular gene or to an mRNA transcript of thatgene and, thereby, inhibits the transcription of that gene and/or thetranslation of that mRNA. The antisense molecules are designed so as tointerfere with transcription or translation of a target gene uponhybridization with the target gene or transcript. Those skilled in theart will recognize that the exact length of the antisenseoligonucleotide and its degree of complementarity with its target willdepend upon the specific target selected, including the sequence of thetarget and the particular bases which comprise that sequence. It ispreferred that the antisense oligonucleotide be constructed and arrangedso as to bind selectively with the target under physiologicalconditions, i.e., to hybridize substantially more to the target sequencethan to any other sequence in the target cell under physiologicalconditions. Based upon SEQ ID NO: 1, or upon allelic or homologousgenomnic and/or cDNA sequences, one of skill in the art can easilychoose and synthesize any of a number of appropriate antisense moleculesfor use in accordance with the present invention. In order to besufficiently selective and potent for inhibition, such antisenseoligonucleotides should comprise at least 10 and, more preferably, atleast 15 consecutive bases which are complementary to the target,although in certain cases modified oligonucleotides as short as 7 basesin length have been used successfully as antisense oligonucleotides(Wagner et al., Nature Biotechnol. 14:840-844, 1996). Most preferably,the antisense oligonucleotides comprise a complementary sequence of20-30 bases. Although oligonucleotides may be chosen which are antisenseto any region of the gene or mRNA transcripts, in preferred embodimentsthe antisense oligonucleotides correspond to N-terminal or 5′ upstreamsites such as translation initiation, transcription initiation orpromoter sites. In addition, 3′-untranslated regions may be targeted.Targeting to mRNA splicing sites has also been used in the art but maybe less preferred if alternative mRNA splicing occurs. In addition, theantisense is targeted, preferably, to sites in which mRNA secondarystructure is not expected (see, e.g., Sainio et al., Cell Mol.Neurobiol. 14(5):439-457, 1994) and at which proteins are not expectedto bind. Finally, although SEQ ID NO: 1 discloses a cDNA sequence one ofordinary skill in the art may easily derive the genomic DNAcorresponding to the cDNA of SEQ ID NO: 1. Thus, the present inventionalso provides for antisense oligonucleotides which are complementary tothe genomic DNA corresponding to SEQ ID NO: 1. Similarly, antisense toallelic or homologous cDNAs and genomic DNAs are enabled without undueexperimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-O-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose instead of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding Smad6polypeptides, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An-expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., luciferase, β-galactosidase or alkaline phosphatase),and genes which visibly affect the phenotype of transformed ortransfected cells, hosts, colonies or plaques (e.g., green fluorescentprotein). Preferred vectors are those capable of autonomous replicationand expression of the structural gene products present in the DNAsegments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Such 5′ non-transcribed regulatory sequences especially willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding Smad6 polypeptide or fragment or variantthereof. That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen), which contains an Epstein Barr virus (EBV) originof replication, facilitating the maintenance of plasmid as a multicopyextrachromosomal element. Another expression vector is the pEF-BOSplasmid containing the promoter of polypeptide Elongation Factor 1α,which stimulates efficiently transcription in vitro. The plasmid isdescribed by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), andits use in transfection experiments is disclosed by, for example,Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferredexpression vector is an adenovirus, described by Stratford-Perricaudet,which is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,1992). The use of the adenovirus as an Adeno.P1A recombinant isdisclosed by Warnier et al., in intradermal injection in mice forimmunization against P1A (Int. J. Cancer, 67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

The invention also permits the construction of Smad6 gene transgenicsand “knock-outs” in cells and in animals, providing materials forstudying certain aspects of TGF-β superfamily signal transduction andthe effects thereof on cellular, developmental and physiologicalprocesses.

The invention also provides isolated polypeptides, which include thepolypeptide of SEQ ID NO: 2 and unique fragments of SEQ ID NO: 2. Smad6polypeptides are encoded by nucleic acids described above, e.g., thosewhich hybridize to SEQ ID NO: 1. Such polypeptides are useful, forexample, alone or as fusion proteins to generate antibodies, as acomponents of an immunoassay.

A unique fragment of a Smad6 polypeptide, in general, has the featuresand characteristics of unique fragments as discussed above in connectionwith nucleic acids. As will be recognized by those skilled in the art,the size of the unique fragment will depend upon factors such as whetherthe fragment constitutes a portion of a conserved protein domain. Thus,some regions of SEQ ID NO: 2 will require longer segments to be unique(e.g., 20, 30, 50, 75, and 100 amino acid long) while others willrequire only short segments, typically between 5 and 12 amino acids(e.g. 5, 6, 7, 8, 9, 10, 11 and 12 amino acids long). Virtually anysegment of SEQ ID NO: 2 which is not overlapped by SEQ ID NO: 5, andthat is 10 or more amino acids in length will be unique. A uniquefragment of a Smad6 polypeptide excludes fragments completely composedof the amino acid sequence of SEQ ID NO: 5 which overlaps SEQ ID NO: 2.A fragment which is completely composed of the sequence of SEQ ID NO: 5is one which does not include any of the amino acids unique to Smad6.

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptides(such as TGF-β superfamily type I receptors) or fragments thereof,selective binding of nucleic acids or proteins, and enzymatic activity.Those skilled in the art are well versed in methods for selecting uniqueamino acid sequences, typically on the basis of the ability of theunique fragment to selectively distinguish the sequence of interest fromnon-family members. A comparison of the sequence of the fragment tothose on known data bases typically is all that is necessary.

The invention embraces variants of the Smad6 polypeptides describedabove. As used herein, a “variant” of a Smad6 polypeptide is apolypeptide which contains one or more modifications to the primaryamino acid sequence of a Smad6 polypeptide. Modifications which create aSmad6 variant can be made to a Smad6 polypeptide 1) to reduce oreliminate an activity of a Smad6 polypeptide, such as binding to TβR-I;2) to enhance a property of a Smad6 polypeptide, such as proteinstability in an expression system or the stability of protein-proteinbinding; or 3) to provide a novel activity or property to a Smad6polypeptide, such as addition of an antigenic epitope or addition of adetectable moiety. Modifications to a Smad6 polypeptide are typicallymade to the nucleic acid which encodes the Smad6 polypeptide, and caninclude deletions, point mutations, truncations, amino acidsubstitutions and additions of amino acids or non-amino acid moieties.Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the Smad6 amino acid sequence.

In general, variants include Smad6 polypeptides which are modifiedspecifically to alter a feature of the polypeptide unrelated to itsphysiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Similarly, certain amino acids can be changed to enhance expression of aSmad6 polypeptide by eliminating proteolysis by proteases in anexpression system (e.g., dibasic amino acid residues in yeast expressionsystems in which KEX2 protease activity is present).

Mutations of a nucleic acid which encode a Smad6 polypeptide preferablypreserve the amino acid reading frame of the coding sequence, andpreferably do not create regions in the nucleic acid which are likely tohybridize to form secondary structures, such a hairpins or loops, whichcan be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant Smad6 polypeptides) which are silent asto the amino acid sequence of the polypeptide, but which providepreferred codons for translation in a particular host. The preferredcodons for translation of a nucleic acid in, e.g., E. coli, are wellknown to those of ordinary skill in the art. Still other mutations canbe made to the noncoding sequences of a Smad6 gene or cDNA clone toenhance expression of the polypeptide. The activity of variants of Smad6polypeptides can be tested by cloning the gene encoding the variantSmad6 polypeptide into a bacterial or mammalian expression vector,introducing the vector into an appropriate host cell, expressing thevariant Smad6 polypeptide, and testing for a functional capability ofthe Smad6 polypeptides as disclosed herein. For example, the variantSmad6 polypeptide can be tested for inhibition of TβR-I (and/or activinor BMP receptor) signalling activity as disclosed in the Examples, orfor inhibition of Smad 1 or Smad2 phosphorylation as is also disclosedherein. Preparation of other variant polypeptides may favor testing ofother activities, as will be known to one of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in Smad6 polypeptides to provide functionallyequivalent variants of the foregoing polypeptides, i.e, variants whichretain the functional capabilities of the Smad6 polypeptides. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution which does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Exemplary functionally equivalentvariants of the Smad6 polypeptides include conservative amino acidsubstitutions of SEQ ID NO: 2. Conservative substitutions of amino acidsinclude substitutions made amongst amino acids within the followinggroups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;(f) Q, N; and (g) E, D.

For example, one can make conservative amino acid substitutions to theamino acid sequence of the Smad6 polypeptide using methods known in theart. Exemplary methods for identifying functional variants of bindingpeptides are provided in a published PCT application of Strominger andWucherpfennig (PCT/US96/03182, describes the identification of variantsof HLA class II binding peptides). The described methods can be used toidentify Smad6 variants which bind TβR-I or other TGF-β superfamilyreceptors or receptor complexes. These variants can be tested, e.g., forimproved stability and are useful, inter alia, in regulation of TGF-βsuperfamily signalling.

Conservative amino-acid substitutions in the amino acid sequence ofSmad6 polypeptides to produce functionally equivalent variants of Smad6polypeptides typically are made by alteration of the nucleic acidencoding Smad6 polypeptides (SEQ ID NO: 1). Such substitutions can bemade by a variety of methods known to one of ordinary skill in the art.For example, amino acid substitutions may be made by PCR-directedmutation, site-directed mutagenesis according to the method of Kunkel(Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemicalsynthesis of a gene encoding a Smad6 polypeptide. Where amino acidsubstitutions are made to a small unique fragment of a Smad6polypeptide, such as a TβR-I binding site peptide, the substitutions canbe made by directly synthesizing the peptide. The activity offunctionally equivalent fragments of Smad6 polypeptides can be tested bycloning the gene encoding the altered Smad6 polypeptide into a bacterialor mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered Smad6 polypeptide, andtesting for a functional capability of the Smad6 polypeptides asdisclosed herein. Peptides which are chemically synthesized can betested directly for function, e.g., for binding to TβR-I.

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof the Smad6 protein molecules (SEQ ID NO: 2). A variety ofmethodologies well-known to the skilled practitioner can be utilized toobtain isolated Smad6 molecules. The polypeptide may be purified fromcells which naturally produce the polypeptide by chromatographic meansor immunological recognition. Alternatively, an expression vector may beintroduced into cells to cause production of the polypeptide. In anothermethod, mRNA transcripts may be microinjected or otherwise introducedinto cells to cause production of the encoded polypeptide. Translationof mRNA in cell-free extracts such as the reticulocyte lysate systemalso may be used to produce polypeptide. Those skilled in the art alsocan readily follow known methods for isolating Smad6 polypeptides. Theseinclude, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography andimmune-affinity chromatography.

The invention also makes it possible isolate proteins such as TβR-I,ActR-IB and BMPR-IB by the binding of such proteins to Smad6 asdisclosed herein. The identification of this binding also permits one ofskill in the art to block the binding of Smad6 to other proteins, suchas TβR-I. For example, binding of such proteins can be affected byintroducing into a biological system in which the proteins bind (e.g., acell) a polypeptide including a Smad6 TβR-I binding site in an amountsufficient to reduce or even block the binding of Smad6 and TβR-I. Theidentification of Smad6 binding to TGF-β superfamily receptors alsoenables one of skill in the art to isolate Smad6 amino acid sequenceswhich bind to such receptors and prepare modified proteins, usingstandard recombinant DNA techniques, which can bind to proteins such asTβR-I, ActR-IB and BMPR-IB. For example, when one desires to target acertain protein to a TβR-I receptor complex, one can prepare a fusionpolypeptide of the protein and the Smad6 TβR-I binding site. Additionaluses are described further herein.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from SEQ ID NO: 2. A dominant negative polypeptideis an inactive variant of a protein, which, by interacting with thecellular machinery, displaces an active protein from its interactionwith the cellular machinery or competes with the active protein, therebyreducing the effect of the active protein. For example, a dominantnegative receptor which binds a ligand but does not transmit a signal inresponse to binding of the ligand can reduce the biological effect ofexpression of the ligand. Likewise, a dominant negativecatalytically-inactive kinase which interacts normally with targetproteins but does not phosphorylate the target proteins can reducephosphorylation of the target proteins in response to a cellular signal.Similarly, a dominant negative transcription factor which binds to apromoter site in the control region of a gene but does not increase genetranscription can reduce the effect of a normal transcription factor byoccupying promoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and using standard mutagenesis techniques tocreate one or more dominant negative variant polypeptides. For example,given the teachings contained herein of a Smad6 polypeptide, one ofordinary skill in the art can modify the sequence of the Smad6polypeptide by site-specific mutagenesis, scanning mutagenesis, partialgene deletion or truncation, and the like. See, e.g., U.S. Pat. No.5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilledartisan then can test the population of mutagenized polypeptides fordiminution in a selected activity (e.g., Smad6 reduction of TGF-βsignalling activity) and/or for retention of such an activity. Othersimilar methods for creating and testing dominant negative variants of aprotein will be apparent to one of ordinary skill in the art.

Dominant negative Smad6 proteins include variants in which a portion ofthe binding site of Smad6 for a TGF-β superfamily receptor has beenmutated or deleted to reduce or eliminate Smad6 interaction with theTGF-β receptor complex. Other examples include Smad6 variants in whichthe ability to inhibit phosphorylation of Smad 1, Smad2 and/or Smad3 isreduced. One of ordinary skill in the art can readily prepare Smad6variants bearing mutations or deletions in the C-terminal domain (e.g.,in the MH2 domain) or in the N-terminal domain (e.g., in theglycine/glutamic acid residue rich region) and test such variants for aSmad6 activity.

The invention also involves agents such as polypeptides which bind toSmad6 polypeptides and to complexes of Smad6 polypeptides and bindingpartners such as TβR-I. Such binding agents can be used, for example, inscreening assays to detect the presence or absence of Smad6 polypeptidesand complexes of Smad6 polypeptides and their binding partners and inpurification protocols to isolate Smad6 polypeptides and complexes ofSmad6 polypeptides and their binding partners. Such agents also can beused to inhibit the native activity of the Smad6 polypeptides or theirbinding partners, for example, by binding to such polypeptides, or theirbinding partners or both.

The invention, therefore, embraces peptide binding agents which, forexample, can be antibodies or fragments of antibodies having the abilityto selectively bind to Smad6 polypeptides. Antibodies include polyclonaland monoclonal antibodies, prepared according to conventionalmethodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to Smad6 polypeptides, and complexes of both Smad6polypeptides and their binding partners. These polypeptides may bederived also from sources other than antibody technology. For example,such polypeptide binding agents can be provided by degenerate peptidelibraries which can be readily prepared in solution, in immobilized formor as phage display libraries. Combinatorial libraries also can besynthesized of peptides containing one or more amino acids. Librariesfurther can be synthesized of peptoids and non-peptide syntheticmoieties (e.g., peptidomimetics).

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe Smad6 polypeptide. This process can be repeated through severalcycles of reselection of phage that bind to the Smad6 polypeptide.Repeated rounds lead to enrichment of phage bearing particularsequences. DNA sequence analysis can be conducted to identify thesequences of the expressed polypeptides. The minimal linear portion ofthe sequence that binds to the Smad6 polypeptide can be determined. Onecan repeat the procedure using a biased library containing insertscontaining part or all of the minimal linear portion plus one or moreadditional degenerate residues upstream or downstream thereof. Yeasttwo-hybrid screening methods also may be used to identify polypeptidesthat bind to the Smad6 polypeptides. Thus, the Smad6 polypeptides of theinvention, or a fragment thereof, can be used to screen peptidelibraries, including phage display libraries, to identify and selectpeptide binding partners of the Smad6 polypeptides of the invention.Such molecules can be used, as described, for screening assays, forpurification protocols, for interfering directly with the functioning ofSmad6 and for other purposes that will be apparent to those of ordinaryskill in the art.

A Smad6 polypeptide, or a fragment thereof, also can be used to isolatetheir native binding partners, including, e.g., the TGF-β receptorcomplex. Isolation of such binding partners may be performed accordingto well-known methods. For example, isolated Smad6 polypeptides can beattached to a substrate (e.g., chromatographic media, such aspolystyrene beads, or a filter), and then a solution suspected ofcontaining the TGF-β receptor complex may be applied to the substrate.If a TGF-β receptor complex which can interact with Smad6 polypeptidesis present in the solution, then it will bind to the substrate-boundSmad6 polypeptide. The TGF-β receptor complex then may be isolated.Other proteins which are binding partners for Smad6, such as otherSmads, and activin or BMP receptor complexes, may be isolated by similarmethods without undue experimentation.

It will also be recognized that the invention embraces the use of theSmad6 cDNA sequences in expression vectors, as well as to transfect hostcells and cell lines, be these prokaryotic (e.g., E. coli), oreukaryotic (e.g., CHO cells, COS cells, yeast expression systems andrecombinant baculovirus expression in insect cells). Especially usefulare mammalian cells such as human, mouse, hamster, pig, goat, primate,etc. They may be of a wide variety of tissue types, and include primarycells and cell lines. Specific examples include keratinocytes,fibroblasts, COS cells, peripheral blood leukocytes, bone marrow stemcells and embryonic stem cells. The expression vectors require that thepertinent sequence, i.e., those nucleic acids described supra, beoperably linked to a promoter.

The isolation of the Smad6 gene also makes it possible for the artisanto diagnose a disorder characterized by aberrant expression of Smad6.These methods involve determining expression of the Smad6 gene, and/orSmad6 polypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes.

The invention further provides methods for reducing or increasing TGF-βsuperfamily signal transduction in a cell. Such methods are useful invitro for altering the TGF-β signal transduction, for example, intesting compounds for potential to block aberrant TGF-β signaltransduction or increase deficient TGF-β signal transduction. In vivo,such methods are useful for modulating growth, e.g., to treat cancer andfibrosis. Increasing TGF-β signal transduction in a cell by, e.g.,introducing a dominant negative Smad6 polypeptide or Smad6 antisenseoligonucleotides in the cell, can be used to provide a model system fortesting the effects of putative inhibitors of TGF-β signal transduction.Such methods also are useful in the treatment of conditions which resultfrom excessive or deficient TGF-β signal transduction. TGF-β signaltransduction can be measured by a variety of ways known to one ofordinary skill in the art, such as the reporter systems described in theExamples. Various modulators of Smad6 activity can be screened foreffects on TGF-β signal transduction using the methods disclosed herein.The skilled artisan can first determine the modulation of a Smad6activity, such as TGF-β signalling activity, and then apply such amodulator to a target cell or subject and assess the effect on thetarget cell or subject. For example, in screening for modulators ofSmad6 useful in the treatment of cancer, cells in culture can becontacted with Smad6 modulators and the increase or decrease of growthor focus formation of the cells can be determined according to standardprocedures. Smad6 activity modulators can be assessed for their effectson other TGF-β signal transduction downstream effects by similar methodsin many cell types.

Thus it can be of therapeutic benefit to administer Smad6 protein ornucleic acid encoding a Smad6 protein, or an agonist or antagonist ofSmad6, to modulate TGF-β superfamily activity in certain conditionscharacterized by abnormal TGF-β superfamily activity. Specific examplesof conditions involving abnormally elevated BMP activity includeossification of the posterior longitudinal ligament (Yonemori et al.,Am. J. Pathol. 150:1335-1347, 1997) and ossification of the ligamentflavum (Hayashi et al., Bone 21:23-30, 1997). Specific examples ofconditions involving abnormal TGF-β activity include liver fibrosisincluding cirrhosis and veno-occlusive disease; kidney fibrosisincluding glomerulonephritis, diabetic nephropathy, allograft rejectionand HIV nephropathy; lung fibrosis including idiopathic fibrosis andautoimmune fibrosis; skin fibrosis including systemic sclerosis,keloids, hypertrophic burn scars and eosinophilia-myalgia syndrome;arterial fibrosis including vascular restenosis and atherosclerosis;central nervous system fibrosis including intraocular fibrosis; andother fibrotic diseases including rheumatoid arthritis andnasalpolyposis. (see, e.g., Border and Noble, N. Engl. J. Med.331:1286-1292, 1994).

An effective amount of Smad6, or an antagonist thereof, is administeredto treat the condition, which amount can be determined by one ofordinary skill in the art by routine experimentation. For example, todetermine an effective amount of Smad6 for treating ossification, Smad6can be administered and the progress of the ossification monitored usingstandard medical diagnostic methods. An amount of Smad6 which reducesthe progression of the ossification, or even halts the progression ofthe ossification is an effective amount. The person of ordinary skill inthe art will be familiar with such methods. Other conditions involvingabnormally elevated or reduced TGF-β superfamily activity can be treatedin a like manner, by administering Smad6 or an agonist thereof, or aSmad6 antagonist, respectively, to reduce or elevate the TGF-βsuperfamily activity into normal ranges as needed. Smad6 antagonistsinclude the antibodies to Smad6, dominant negative variants of Smad6 andatitisense Smad6 nucleic acids described above. Smad6 agonists includeagents which increase Smad6 expression, binding or activity.

When administered, the therapeutic compositions of the present inventionare administered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines and optionally other therapeutic agents.

The therapeutics of the invention can be administered by anyconventional route, including injection or by gradual infusion overtime. The administration may, for example, be oral, intravenous,intraperitoneal, intramuscular, intracavity, subcutaneous, ortransdermal. When antibodies are used therapeutically, a preferred routeof administration is by pulmonary aerosol. Techniques for preparingaerosol delivery systems containing antibodies are well known to thoseof skill in the art. Generally, such systems should utilize componentswhich will not significantly impair the biological properties of theantibodies, such as the paratope binding capacity (see, for example,Sciarra and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences,18th edition, 1990, pp 1694-1712; incorporated by reference). Those ofskill in the art can readily determine the various parameters andconditions for producing antibody aerosols without resort to undueexperimentation. When using antisense preparations of the invention,slow intravenous administration is preferred.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a pharmaceutical preparation thatalone, or together with further doses, produces the desired response.For example, in the case of treating cancer, the desired response isinhibiting the progression of the cancer. In the case of treatingossification of the ligamentum flavum, the desired response isinhibiting the progression of the ossification. This may involve onlyslowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. This can be monitored by routine methods or can bemonitored according to diagnostic methods of the invention discussedherein.

The invention also contemplates gene therapy. The procedure forperforming ex vivo gene therapy is outlined in U.S. Pat. No. 5,399,346and in exhibits submitted in the file history of that patent, all ofwhich are publicly available documents. In general, it involvesintroduction in vitro of a functional copy of a gene into a cell(s) of asubject which contains a defective copy of the gene, and returning thegenetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permitexpression of the gene in the genetically engineered cell(s). Numeroustransfection and transduction techniques as well as appropriateexpression vectors are well known to those of ordinary skill in the art,some of which are described in PCT application WO95/00654. In vivo genetherapy using vectors such as adenovirus, retroviruses, herpes virus,and targeted liposomes also is contemplated according to the invention.

The invention further provides efficient methods of identifyingpharmacological agents or lead compounds for agents active at the levelof a Smad6 or Smad6 fragment modulatable cellular function. Inparticular, such functions include TGF-β, activin and BMP signaltransduction and formation of a TGF-β superfamily receptor-Smad6 proteincomplex. Generally, the screening methods involve assaying for compoundswhich interfere with a Smad6 activity such as TGF-β receptor-Smad6binding, etc. Such methods are adaptable to automated, high throughputscreening of compounds. The target therapeutic indications forpharmacological agents detected by the screening methods are limitedonly in that the target cellular function be subject to modulation byalteration of the formation of a complex comprising a Smad6 polypeptideor fragment thereof and one or more natural Smad6 intracellular bindingtargets, such as TGF-β receptor. Target indications include cellularprocesses modulated by TGF-β, activin and BMP signal transductionfollowing receptor-ligand binding.

A wide variety of assays for pharmacological agents are provided,including, labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays, cell-based assayssuch as two- or three-hybrid screens, expression assays, etc. Forexample, three-hybrid screens are used to rapidly examine the effect oftransfected nucleic acids on the intracellular binding of Smad6 or Smad6fragments to specific intracellular targets. The transfected nucleicacids can encode, for example, combinatorial peptide libraries orantisense molecules. Convenient reagents for such assays, e.g., GAL4fusion proteins, are known in the art. An exemplary cell-based assayinvolves transfecting a cell with a nucleic acid encoding a Smad6polypeptide fused to a GAL4 DNA binding domain and a nucleic acidencoding a TGF-β receptor domain which interacts with Smad6 fused to atranscription activation domain such as VP16. The cell also contains areporter gene operably linked to a gene expression regulatory region,such as one or more GAL4 binding sites. Activation of reporter genetranscription occurs when the Smad6 and TGF-β receptor fusionpolypeptides bind such that the GAL4 DNA binding domain and the VP16transcriptional activation domain are brought into proximity to enabletranscription of the reporter gene. Agents which modulate a Smad6polypeptide mediated cell function are then detected through a change inthe expression of reporter gene. Methods for determining changes in theexpression of a reporter gene are known in the art.

Smad6 fragments used in the methods, when not produced by a transfectednucleic acid are added to an assay mixture as an isolated polypeptide.Smad6 polypeptides preferably are produced recombinantly, although suchpolypeptides may be isolated from biological extracts. Recombinantlyproduced Smad6 polypeptides include chimeric proteins comprising afusion of a Smad6 protein with another polypeptide, e.g., a polypeptidecapable of providing or enhancing protein-protein binding, sequencespecific nucleic acid binding (such as GAL4), enhancing stability of theSmad6 polypeptide under assay conditions, or providing a detectablemoiety, such as green fluorescent protein or Flag epitope as provided inthe examples below.

The assay mixture is comprised of a natural intracellular Smad6 bindingtarget such as a TGF-β receptor or fragment thereof capable ofinteracting with Smad6. While natural Smad6 binding targets may be used,it is frequently preferred to use portions (e.g., peptides or nucleicacid fragments) or analogs (i.e., agents which mimic the Smad6 bindingproperties of the natural binding target for purposes of the assay) ofthe Smad6 binding target so long as the portion or analog providesbinding affinity and avidity to the Smad6 fragment measurable in theassay.

The assay mixture also comprises a candidate pharmacological agent.Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate pharmacological agents aresmall organic compounds, i.e., those having a molecular weight of morethan 50 yet less than about 2500, preferably less than about 1000 and,more preferably, less than about 500. Candidate agents comprisefunctional chemical groups necessary for structural interactions withpolypeptides and/or nucleic acids, and typically include at least anamine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups and more preferably at least three of thefunctional chemical groups. The candidate agents can comprise cycliccarbon or heterocyclic structure and/or aromatic or polyaromaticstructures substituted with one or more of the above-identifiedfunctional groups. Candidate agents also can be biomolecules such aspeptides, saccharides, fatty acids, sterols, isoprenoids, purines,pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the agent is a nucleic acid,the agent typically is a DNA or RNA molecule, although modified nucleicacids as defined herein are also contemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs of the agents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease, inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidatepharmacological agent, the Smad6 polypeptide specifically binds thecellular binding target, a portion thereof or analog thereof. The orderof addition of components, incubation temperature, time of incubation,and other perimeters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe Smad6 polypeptide and one or more binding targets is detected by anyconvenient method available to the user. For cell free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximum signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromatographic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. The transcript resulting from areporter gene transcription assay of Smad6 polypeptide interacting witha target molecule typically encodes a directly or indirectly detectableproduct, e.g., β-galactosidase activity, luciferase activity, and thelike. For cell free binding assays, one of the components usuallycomprises, or is coupled to, a detectable label. A wide variety oflabels can be used, such as those that provide direct detection (e.g.,radioactivity, luminescence, optical or electron density, etc). orindirect detection (e.g., epitope tag such as the FLAG epitope, enzymetag such as horseradish peroxidase, etc.). The label may be bound to aSmad6 binding partner, or incorporated into the structure of the bindingpartner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The invention provides Smad6-specific binding agents, methods ofidentifying and making such agents, and their use in diagnosis, therapyand pharmaceutical development. For example, Smad6-specificpharmacological agents are useful in a variety of diagnostic andtherapeutic applications, especially where disease or disease prognosisis associated with improper utilization of a pathway involving Smad6,e.g., TGF-β induced phosphorylation of Smad1 or Smad2, TGF-β superfamilyreceptor-Smad6 complex formation, etc. Novel Smad6-specific bindingagents include Smad6-specific antibodies and other natural intracellularbinding agents identified with assays such as two hybrid screens, andnon-natural intracellular binding agents identified in screens ofchemical libraries and the like.

In general, the specificity of Smad6 binding to a binding agent is shownby binding equilibrium constants. Targets which are capable ofselectively binding a Smad6 polypeptide preferably have bindingequilibrium constants of at least about 10⁷ M⁻¹, more preferably atleast about 10⁸ M⁻¹, and most preferably at least about 10⁹ M⁻¹. Thewide variety of cell based and cell free assays may be used todemonstrate Smad6-specific binding. Cell based assays include one, twoand three hybrid screens, assays in which Smad6-mediated transcriptionis inhibited or increased, etc. Cell free assays include Smad6-proteinbinding assays, immunoassays, etc. Other assays useful for screeningagents which bind Smad6 polypeptides include fluorescence resonanceenergy transfer (FRET), and electrophoretic mobility shift analysis(EMSA).

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a retrovirus, or othervirus; a liposome) can have a targeting molecule attached thereto. Forexample, a molecule such as an antibody specific for a surface membraneprotein on the target cell or a ligand for a receptor on the target cellcan be bound to or incorporated within the nucleic acid deliveryvehicle. For example, where liposomes are employed to deliver thenucleic acids of the invention, proteins which bind to a surfacemembrane protein associated with endocytosis may be incorporated intothe liposome formulation for targeting and/or to facilitate uptake. Suchproteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

EXAMPLES Methods

Cloning of Mouse Smad6 and Northern Blot

A mouse lung cDNA library (Stratagene, La Jolla, Calif.) was screenedwith an EST clone (clone ID 429356) as a probe. One of the clonescontained the entire coding region of the mouse Smad6 and was sequencedusing an ALFred sequencer (Pharmacia Biotech) and a Sequenase sequencingkit (USB, Cleveland, Ohio). Sequence analysis was done with DNASTAR(DNASTAR, Inc.). Human and mouse tissue blots (Clontech, Palo Alto,Calif.) were probed with the EST clone.

Plasmids

Mammalian expression vectors with an amino-terminal tag (FLAG or Myc)were constructed by inserting oligonucleotides encoding the epitope-tagsequence into pcDNA3 (Invitrogen). The coding region of the mouse Smad6was amplified by PCR and subcloned into Myc-pcDNA3 or FLAG-pcDNA3. Theintegrity of the products were confirmed by sequencing. Smad1, Smad2,Smad3 and Smad4 expression plasmids were constructed in a similarmanner.

Affinity Cross-linking and Immunoprecipitation

Iodination of TGF-β1 (R&D Systems), activin A (gift of Y. Eto), andOP1/BMP-7 (gift of T. K. Sampath) and the following immunoprecipitationwere performed as described (Okadome et al., J. Biol. Chem.271:21687-21690, 1996).

Western Blot and in vivo Phosphorylation

COS-7 cells were transiently transfected using DMRIE-C (Gibco/BRL,Gaithersburg, Md.). [³²P]orthophosphate- or[³⁵]methionine/cysteine-labeling and immunoprecipitation were done asdescribed by Nakao et al. (J. Biol. Chem. 272:2896-2900, 1997). ForWestern blot of the immunoprecipitated proteins, tagged proteins weredetected by chemiluminescence (ECL, Amersham, Arlington Heights, Ill.).

Luciferase Assays

Mink R mutant cells were transiently transfected with an appropriatecombination of a reporter, expression plasmids, and pcDNA3 using Tfx-50(Promega). Total amounts of transfected DNA were the same in eachexperiment, and values were normalized using sea pansy luciferaseactivity under the control of the thymidine kinase promoter (pRL-TK,Toyo Ink).

Cell Cultures

C1C12 cells, F9 cells, and ST2 cells were obtained from Riken Cell Bank(Tsukuba, Japan). 10T1/2 cells were from American Type CultureCollection (Bethesda, Md., USA). C1C12 cells and 10T1/2 cells werecultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, 100units of penicillin and 50 μg of streptomycin per ml. F9 cells werecultured in DMEM with 15% FBS and the antibiotics, and ST2 cells werecultured in RPMI1640 with 10% FBS and the antibiotics. The cells werekept in 5% CO₂ humid atmosphere at 37° C.

Poly(A)⁺ RNA Isolation and Northern Blotting

Poly(A⁺) RNA was obtained using Oligotex dT-30 Super latex beads (TakaraShuzo Co., Ltd.) according to the manufacturer's method. Poly(A)⁺ RNA (3μg) from cells treated with BMP-2 (300 ng/ml), BMP-7/OP-1(300 ng/ml), orTGF-β1 (25 ng/ml) for various time periods were electrophoresed in 1%gel in the presence of 2.2 M formaldehyde gels and blotted to Hybond Nmembranes (Amersham). The complete coding region of mouse Smad6 cDNA waslabeled by [α-³²P]dCTP using Random Primer Labeling Kit (Takara ShuzoCo., Ltd.). Hybridization was performed in a solution containing 5×SSC,1% SDS, 5×Denhardt's solution and 10 μg/ml salmon sperm DNA at 65° C.with 2×SSC, 1% SDS for 20 min. twice, 0.5×SSC, 1% SDS for 30 min.0.2×SSC, 1% SDS for 10 min. The filters were stripped by boileddistilled water containing 0.1% SDS and rehybridized.

Example 1 Identification of Smad6 and Determination of its Expression

In search of new members of the Smad family, several expressed-sequencetag (EST) sequences that are not ascribed to the five mammalian Smadscharacterized previously (Massagúe et al., 1997) were identified. Wescreened a mouse lung cDNA library using one of the EST clones (clone ID429356) as a probe and identified overlapping clones encoding the sameprotein of 495 amino acids with a predicted molecular weight of 53.7 kDa(FIG. 1 a, SEQ ID NO: 2; GenBank accession number AF010133). Identicalresidues are boxed. The C-terminal amino acid sequence of the proteinwas almost identical with the entire protein sequence of a GenBank clonedeposited as the human Smad6 (235 amino acids, accession number:U59914). It thus was concluded that our clone is the full-length mouseSmad6. Chromosomal localization of the human Smad6 (JV15-1) has beenreported (Riggins et al., Nature Genet. 13:347-349, 1996). Except forthe human Smad6, all of the Smads, including Drosophila and C. elegansSmads, comprise conserved N-terminal and C-terminal regions (MH1 andMH2, respectively) separated by a proline-rich linker region of variablelength and sequence, although Smad4 has a unique insert in its MH2region (Massagúe, et al., 1997). The C-terminal one-third of Smad6shares the conserved sequence with the MH2 regions of the other Smads,whereas its N-terminal region shows a striking difference from theconserved MH1 sequence (FIG. 1 a), suggesting a novel function of thismolecule.

Northern blot analysis of various human and mouse tissues, using thehuman EST clone 429356 as a probe, revealed relatively ubiquitousexpression of the mRNA species of 3.0 kb with the highest expression inlung (FIG. 1 b; human (left) and mouse (right)).

Example 2 Receptor Binding of Smad6

Members of the TGF-β superfamily exert their diverse effects throughbinding to two types of receptors with serine-threonine kinase activity(Yingling et al., Biochim. Biophys. Acta 1242:115-136, 1995). The ligandfirst binds to the type II receptor, which consequently activates thetype I receptor by direct phosphorylation. The activated type I receptorthen phosphorylates ligand-specific Smads such as Smad1, Smad2, andSmad3 (Massagúe, et al., 1997; Zhang et al., Nature 383:168-172, 1996;Lagna et al., Nature 383:832-836, 1996; MacíasSilva et al., Cell87:1215-1224, 1996). The association of Smad2 with TβR-I requiresactivation of TβR-I requires activation of TβR-I by the type II receptor(TβR-II) (MacíasSilva et al., 1996). Smad2, however, interacts withTβR-I only transiently under physiological conditions, since Smad2 isreleased from TβR-I after phosphorylation by the receptor. Theinteraction of Smad2 with the TβR-I has thus been observed only when thekinase-defective form of TβR-I is used (Macías-Silva et al., 1996). Itshould be noted that Smad4 does not associate the receptors (Zhang etal., 1996).

The interaction of Smad6 with the type I receptors was examined inaffinity cross-linking assays. COS-7 cells were transfected withFLAG-tagged Smad6 (F-Smad6) or FLAG-tagged Smad2 (F-Smad2) incombination with the wild type (wt) or kinase-defective (KR) HA-taggedTβR-I and hexahistidine-tagged TβR-II. Cells were affinity labeled with¹²⁵I-TGF-β1 and lysates were immunoprecipitated with anti-HA antibody oranti-FLAG M2 antibody. Immune complexes were subjected to SDS-PAGE andautoradiography. Smad6 bound to both wild type and kinase-defectiveTβR-I depending on the kinase activity of TβR-II. In contrast, Smad2bound to kinase-defective TβR-I but not to the wild type TβR-I. Smad6bound to the TGF-β receptor complexes as revealed by the coprecipitationof the receptor complexes with Smad6 (FIG. 2 a). Similarly to Smad2, thebinding of Smad6 to TβR-I required the kinase activity of TβR-II (FIG. 2a). Smad6, however, stably bound to the wild type TβR-I.

Similar results were obtained with the activin type IB receptor(ActR-IB) using ¹²⁵I-activin A (FIG. 2B) and the BMP type IB receptor(BMPR-IB) using ¹²⁵I-OP-1/BMP-7 (FIG. 2C) in which Smad6 bound to bothwild type and kinase-defective type I receptors. These results suggestthat Smad6 binds to the type I receptors in a ligand-dependent mannerbut exerts a role different from that of the other Smads.

Example 3 Effect of Smad6 on Phosphorylation of Smads

Smad2 is phosphorylated at its carboxy-terminal end by activated TβR-I(Macías-Silva et al., 1996). The phosphorylation is essential to thefollowing downstream signaling events that culminate in transcriptionalactivation of the target genes, since disruption of the phosphorylationsites abrogated TGF-β-induced responses (MacíasSilva et al., 1996). Thusthe effect of Smad6 on the phosphorylation of Smad2 was examined (FIG.3). COS-7 cells were transiently transfected with constitutively active(TD) TβR-I, FLAG-Smad2 (F-Smad2), and/or Myc-Smad6 (M-Smad6). Cells werelabeled with [³²P]orthophosphate and lysates were subjected toimmunoprecipitation with anti-Myc antibody. Phosphorylated Smad6 wasdetected by SDS-PAGE and autoradiography. Doublet bands ofphosphorylated Smad6 were detected. Cell lysates also wereimmunoprecipitated with anti-FLAG antibody to detect Smad2phosphorylation. Expression levels of Smad6 (panel A), Smad2 and TβR-I(TD) (panel B) were monitored by labeling of the cells with[³⁵S]methionine/cysteine.

Neither TβR-I (TD) or FLAG-Smad2 affected the phosphorylation of Smad6.Phosphorylation of Smad2 induced by constitutively active TβR-I wassuppressed by Smad6 (39% reduction as normalized for the ³⁵S-labeledband), whereas Smad2 did not affect constitutive phosphorylation ofSmad6 (FIGS. 3A, B).

Smad3 and Smad2 share 91% identity in their amino acid sequences and areindependently shown to mediate TGF-β signals (Eppert et al. Cell86:543-552, 1996; Zhang et al., 1996), although functional differencesof the two molecules are still unknown. A similar experiment as abovewas done with Smad3. Smad6 rather enhanced receptor-inducedphosphorylation of Smad3 (FIG. 3C), suggesting differential effects ofSmad6 to these closely related molecules.

Next, the effect of Smad6 on Smad1 phosphorylation was studied (FIG.3D). Smad1 was phosphorylated by the constitutively active BMP type IAreceptor (BMPR-IA) as well as BMPR-IB. Smad6 efficiently inhibitedphosphorylation induced by the latter (60% reduction) but not by theformer. These results suggest that Smad6 acts as an inhibitor to certainmembers of the Smad family.

Example 4 Effect of Smad6 on Smad Complex Formation

Smad2 heteromerizes with Smad4 upon phosphorylation by TβR-I (Lagna etal., 1996). It was recently shown that TGF-β also induces association ofSmad2 and Smad3 (Nakao et al., EMBO. J. 16:5353-5362, 1997). The effectof Smad6 on the heteromerization of these Smads was examined (FIG. 4).COS-7 cells were transfected with the indicated combination of plasmidsand subjected to immunoprecipitation followed by Western blot detection.Expression levels of Smad2 (bottom), Smad4 (middle), and Smad6 (middle)were monitored. Note that Smad6 did not interact with Smad2 under theseconditions (top).

Smad2 formed a complex with Smad4 in the presence of constitutivelyactive TβR-I as shown by coprecipitation of Smad4 with Smad2. Thecomplex formation was abrogated by Smad6 (FIG. 4A, top panel).TβR-I-induced interaction of Smad3 and Smad4, however, was not affectedby Smad6 (FIG. 4B), as Smad4 coprecipitated with Smad3 both in thepresence and absence of Smad6 (top). This is consistent with the resultthat Smad6 does not inhibit Smad3 phosphorylation (FIG. 3C).Furthermore, heteromerization of Smad2 and Smad3 was inhibited by Smad6(FIG. 4C), suggesting that phosphorylation of both proteins is necessaryfor this interaction. These results suggest that Smad6 specificallyinterferes with the activation of Smad2 in TGF-β signaling.

Example 5 Effect of Smad6 on TGF-β Signaling

The role of Smad6 in TGF-β signaling was tested, as assessed byluciferase reporter gene assays. P3TP-Lux, a sensitive reporter forTGF-β, was used in R mutant mink cells deficient in TβR-I (FIG. 5). Wildtype TβR-I restored TGF-β response in these cells. Mink R mutant cellsdeficient in TβR-I were transfected with p3TP-Lux reporter, TβR-I, andincreasing amounts (μg) of Smad6 DNA. Cells were treated with (closedbars) or without (open bars) 5 ng/ml TGF-β1 for 24 h. Smad6 suppressedthe activation of the reporter gene in a dose-dependent manner (FIG. 5a). Transcriptional activation by constitutively active TβR-I wassuppressed as well (FIG. 5 b).

Cyclin A expression is necessary for cell cycle progression and issuppressed by TGF-β (Feng et al., J. Biol. Chem. 270:4237-24245, 1995).A cyclin A luciferase reporter, pCAL2, was used to examine the effect ofSmad6 on TGF-β signaling. TβR-I (consitutively active) downregulatedcyclin A luciferase activity, but increasing amounts of Smad6counteracted the effects of TβR-I in the cyclin A luciferase assay (FIG.5 c). These results indicate that Smad6 interfered with TGF-β signals intwo distinct responses.

Example 6 Regulation of Smad6 Expression by TGF-β1 and BMPs

To determine the mechanisms by which the expression of Smad6 iscontrolled, the effect of TGF-β and other family members on Smad6 mRNAexpression was examined. To determine the effect of BMP-2, BMP-2 wasadded at 300 ng/ml to the culture medium of several BMP-2-responsive clllines: F9, C3H10T/½, ST2 and C2C12. After 6 hours, poly A⁺ mRNA wasisolated from the cells as described above. Samples of mRNA wereelectrophoresed, blotted and probed with ³²P-labeled Smad6 codingregion. The results of the Northern blot are shown in FIG. 6A.Expression of Smad6 was induced in all of the BMP-2 responsive celllines after BMP treatment.

To determine the effect of OP-1/BMP-7, the expression of Smad6 mRNA inC2C12 myoblasts after stimulation with 300 ng/ml OP-1/BMP-7 was tested.Poly A⁺ mRNA was isolated at the indicated times following OP-1/BMP-7stimulation. As shown in FIG. 6B, Smad6 expression was induced byOP-1/BMP-7 at 6 hours after stimulation, and remained induced up to atleast 48 hours after stimulation, the duration of the experiment.

To determine the effect of TGF-β1, the expression of Smad6 mRNA in C2C12myoblasts after stimulation with 25 ng/ml TGF-β1 was tested. Poly A⁺mRNA was isolated at the indicated times following TGF-β1 stimulation.As shown in FIG. 6C, Smad6 expression was induced by TGF-β1 at 1 hourafter stimulation, but decreased thereafter. After 12 hours, theexpression of Smad6 mRNA was below the basal level of expression.

In summary, the BMPs tested (BMP-2 and BMP-7/OP-1) induced theexpression of Smad6 mRNA. In contrast, TGF-β1 initially induced theexpression of Smad6, but Smad6 levels subsequently decreased to belowbasal levels. Thus different members of the TGF superfamily exertopposing effects on the expression of Smad6.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

1. A method for identifying compounds for a pharmacological agent usefulin the diagnosis or treatment of disease associated with Smad6 TGF-βsuperfamily inhibitory activity, wherein said Smad6 (a) is encoded bythe complement of nucleic acid molecules which hybridize under stringentconditions to a molecule consisting of the nucleic acid sequence of SEQID NO: 1 and which code for a polypeptide which inhibits TGF-βsuperfamily signaling, provided that the nucleic acid molecules excludesequences consisting only of SEQ ID NO: 4, wherein the stringentconditions are selected from the group of (1) hybridization at 65° C. inhybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄ (pH 7), 0.5%SDS, 2 mM EDTA; wherein SSC is 0.15 M sodium chloride/0.015 M sodiumcitrate, pH 7, SDS is sodium dodecyl sulphate, and EDTA isethylenediaminetetracetic acid); and (2) hybridization at 65° C. in5×SSC, 1% SDS, 5× Denhardt's solution, and 10 μg/ml salmon sperm DNA (b)is encoded by nucleic acid molecules that differ from the nucleic acidmolecules of (a) in codon sequence due to the degeneracy of the geneticcode, or (c) consists of the amino acid sequence of SEQ ID NO: 2, saidmethod comprising forming a mixture comprising a Smad6 polypeptide, areceptor or receptor complex for a TGF-β superfamily member, and acandidate pharmacological agent, incubating the mixture under conditionswhich, in the absence of the candidate pharmacological agent, permit afirst amount of specific binding of the TGF-β superfamily receptor orreceptor complex by the Smad6 potypeptide, and detecting a second testamount of the specific binding of the TGF-β superfamily receptor orreceptor complex by the Smad6 polypeptide, wherein reduction of thesecond test amount of specific binding relative to the first amount ofspecific binding indicates that the candidate pharmacological agent is acompound for a pharmacological agent which disrupts the Smad6 TGF-βsuperfamily signal transduction inhibitory activity, and whereinincrease of the test amount of specific binding relative to the firstamount of specific binding indicates that the candidate pharmacologicalagent is a compound for a pharmacological agent which enhances the Smad6TGF-β superfamily signal transduction inhibitory activity.
 2. The methodof claim 1, wherein the member of the TGF-β superfamily is selected fromthe group consisting of TGF-β1, activin, BMP-4 and BMP-7.
 3. A methodfor identifying compounds for a pharmacological agent useful in thediagnosis or treatment of disease associated with Smad6 TGF-βsuperfamily signal transduction inhibitory activity, wherein said Smad6(a) is encoded by the complement of nucleic acid molecules whichhybridize under stringent conditions to a molecule consisting of thenucleic acid sequence of SEQ ID NO: 1 and which code for a polypeptidewhich inhibits TGF-β superfamily signaling, provided that the nucleicacid molecules exclude sequences consisting only of SEQ ID NO: 4,wherein the stringent conditions are selected from the group of (1)hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll,0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH₂PO₄(pH 7), 0.5% SDS, 2 mM EDTA; wherein SSC is 0.15 M sodium chloride/0.015M sodium citrate, pH 7, SDS is sodium dodecyl sulphate, and EDTA isethylenediaminetetracetic acid); and (2) hybridization at 65° C. in5×SSC, 1% SDS, 5× Denhardt's solution, and 10 μg/ml salmon sperm DNA (b)is encoded by nucleic acid molecules that differ from the nucleic acidmolecules of (a) in codon sequence due to the degeneracy of the geneticcode, or (c) consists of the amino acid sequence of SEQ ID NO: 2, saidmethod, comprising forming a mixture comprising a Smad6 polypeptide, areceptor or receptor complex for a TGF-β superfamily member, a pathwayrestricted Smad polypeptide and a candidate pharmacological agent,incubating the mixture under conditions which, in the absence of thecandidate pharmacological agent, permit a first amount of TGF-βsuperfamily member induced phosphorylation of the pathway restrictedSmad polypeptide, and detecting a second test amount of TGF-βsuperfamily member induced phosphorylation of the pathway restrictedSmad polypeptide, wherein reduction of the second test amount of thephosphorylation of the pathway restricted Smad polypeptide relative tothe first amount of the phosphorylation of the pathway restricted Smadpolypeptide indicates that the candidate pharmacological agent is acompound for a pharmacological agent which enhances the TGF-βsuperfamily signal transduction inhibitory activity of Smad6 and whereinincrease of the second test amount of the phosphorylation of the pathwayrestricted Smad polypeptide relative to the first amount of thephosphorylation of the pathway restricted Smad polypeptide indicatesthat the candidate pharmacological agent is a lead compound for apharmacological agent which disrupts the TGF-β superfamily signaltransduction inhibitory activity of Smad6.
 4. The method of claim 3,wherein the pathway restricted Smad polypeptide is selected from thegroup consisting of Smad1 and Smad2.
 5. The method of claim 3, whereinthe member of the TGF-β superfamily is selected from the groupconsisting of TGF-β1, activin, BMP-4 and BMP-7.